The present invention relates to the field of techniques for analyzing, separating and purifying species, according to which it is necessary to migrate these species in a fluid known as the “separating medium”.
It is also more particularly directed toward proposing a surface treatment solution which is of use in significantly reducing the nonspecific adsorption of species contained in this fluid to the walls of a channel or of a container containing said fluid.
According to a first aspect, the invention is more particularly directed toward proposing a separating medium that is suitable for separating species in channels or capillaries, at least one of the dimensions of which is submillimetric, and typically between 1 μm and 200 μm, for example 20 μm and 200 μm (referred to hereinbelow as microchannels). The invention in particular concerns methods for separating or analyzing biological macromolecules by capillary electrophoresis, by chromatography or by any method used in microchannels (capillary electrophoresis and capillary chromatography, microfluid systems, and “lab-on-chips”). The invention is particularly useful in the case of electrophoresis.
According to a second aspect the invention also relates more particularly to techniques for analyzing or for separating species, according to which it is necessary to transport said species in a channel, while at the same time minimizing the nonspecific interactions of said species, or of other components of said medium, with the walls of said channel or more generally with walls or solid elements present in said channel or said medium. These are in particular methods for separating or for analyzing biological macromolecules by capillary electrophoresis, by chromatography or by any method carried out in microchannels (microfluid systems, “laboratories on chips”). Examples of such systems are described, for example, in “Capillary electrophoresis in analytical biotechnology”. Righetti ed., CRC press, 1996, or in J. Cheng et al. (1996), Molecular Diagnosis, 1, 183-200. The invention is of particular use in the case of electrophoresis.
The invention also relates to “hybridization” or “affinity” techniques in which the aim is to analyze, within a channel or a container, the species contained in a sample, as a function of their specific affinity for ligands contained in said channel or container, or attached to the walls of said container or said channel, at predetermined positions.
In the context of the invention, the term “nonspecific adsorption” is intended to be used as normally accepted by those skilled in the art, as an interaction of attraction between certain species or impurities contained in a sample and the walls of the container and of the channel which depends weakly or in an insufficiently controlled manner on the characteristics of said species or impurities. In the remainder of the text, the term “adsorption” or “nonspecific adsorption” will be used indifferently to denote the latter, as opposed to an interaction of specific affinity. The term “affinity” is intended to mean an interaction between a species and a substrate, the strength of which depends strongly on said species and on said substrate, and which, in any event, is sufficient to induce the separation or the identification of various species as a function of their biological or physicochemical characteristics.
For the purpose of the invention, the terms “solution for treating” and “treatment solution” are equivalent to the term “medium for treating”.
In the text herein below, the expression “microfluid system” will denote any system in which fluids and/or species contained in a fluid are moved inside a channel or a set of channels, at least one of the dimensions of which is submillimetric, and the term “capillary electrophoresis (CE)” will denote microfluid systems in which the transportation of species is performed by the action of an electric field.
CE and microfluid systems allow faster separations with higher resolutions than the older methods of gel electrophoresis, do not require an anticonvective medium, and their properties have been used widely to perform separations of ions in liquid medium. At the present time, the vast majority of separations of biological macromolecules performed by CE use solutions of interlocked linear water-soluble polymers that have the advantage of being able to be replaced as often as necessary.
Many non-crosslinked polymers have been proposed as media for separating species inside a channel, in particular in the context of capillary electrophoresis. The choice of the best polymer for a given application depends on several parameters. For example, for the separation of analytes as a function of their sizes, it is necessary for the medium to present the analytes with sufficiently resistant topological obstacles (Viovy et al., Electrophoresis, 1993, 14, 322). This involves the separating medium being highly interlocked, and thus relatively viscous. It is also necessary for the polymers present in the separating medium not to undergo any interactions of attraction with the analytes. The reason for this is that interactions of this type give rise to a slowing-down of certain analytes, and to additional dispersion (H. Zhou et al. HPCE 2000, Saarbrucken, 20-24 Feb. 2000). Thus, it is well known that for DNA sequencing, or for protein separation, poorer results are obtained when the matrix has a more hydrophobic nature.
It has also been proposed in the literature to use copolymers as separating medium. In Menchen, WO 94/07133, it is proposed to use as separating medium in capillary electrophoresis, media comprising copolymers of block copolymer type which are said to be “regular” since they have hydrophilic segments of a selected and essentially uniform length and a plurality of regularly spaced hydrophobic segments, at a concentration higher than the overlap concentration between polymers. These media have the advantage of being shear-thinning, i.e. they can be introduced into a capillary under high pressure, while at the same time presenting solid topological obstacles in the absence of external pressure. Unfortunately, the media that may be used according to this principle are difficult to synthesize, which makes them expensive and limits the type of structures that may be envisaged. Also, these polymers are relatively hydrophobic, and their performance qualities for DNA sequencing, for example, are mediocre.
It has also been proposed to use as separating media thermosensitive media, the viscosity of which varies greatly during an increase in temperature. This type of medium has the advantage of allowing the injection of said medium into the capillary at a first temperature in a state of low viscosity, and the separation at a second temperature in a state of higher viscosity that displays good separation efficiency, as is commonly performed in gel electrophoresis, in particular with agarose. Patent applications WO 94/10561 and WO 95/30782 especially propose media that allow an easier injection by raising the temperature. In point of fact, said patent applications essentially describe microgels capable of decreasing in volume at high temperature (thus leading to a dilute solution of discontinuous particles of low viscosity) and of swelling at low temperature until they entirely fill the separating channel (thus giving the medium a gelled nature and good separating properties). Patent application WO 98/10274 itself proposes a molecular separating medium comprising at least one type of block copolymers that is in solution at a first temperature and in a gel-type state at a second temperature. The media described comprise triblock polymers of low molecular masses (typically less than 20,000), of the polyoxyethylene-polyoxypropyiene-polyoxyethylene (POE-Pop-POE) family and more specifically (POE99-POP69-POE99 in which the indices represent the number of monomers of each block) (trade name “Pluronic F127”). At low temperature, the two POE segments at the ends of the triblock systems are water-soluble and, given the low molecular mass of the copolymer, the solutions are relatively nonviscous up to a high concentration. By raising the temperature by about 15-25° C., the central POP segment becomes more hydrophobic, and these polymers become associated to form a gel-type state. However, this mechanism presents several drawbacks in electrophoresis. Firstly, it gives rise to a gel state that has good electrophoretic separating properties only at high polymer concentrations, of greater than 15 g/100 ml or even 20 g/100 ml, which leads to high friction and long migration times. Moreover, the dependence of the properties as a function of the rate of change of temperature makes the reproducibility of the results random. Finally, for many applications and in many devices, it is inconvenient, or even impossible, to change the temperature between the stage of filling of the channel and the separating stage.
In Madabhushi, U.S. Pat. No. 5,552,028, WO 95/16910 and 20 WO 95/16911, it is also proposed to use separating media comprising a screening medium and a surface-interaction component consisting of a polymer with wall-adsorption properties, with a molecular mass of between 5,000 and 1,000,000, of the disubstituted acrylamide polymer type. These matrices, and more particularly polydimethylacrylamide (PDMA), make it possible to reduce the electroosmosis and in certain applications, for instance sequencing, lead to good separating properties. However, they are relatively hydrophobic, which limits their performance qualities for certain applications, for instance DNA sequencing, and is even more harmful for other applications, for instance protein separation. Moreover, they lead to slow separations.
Consequently, despite the large number of studies and systems proposed, a medium that is optimum for all the various aspects of cost, of separation efficiency, of reduction of interactions with the walls and of convenience of use is not available at the present time for all the applications mentioned above.
In connection to uses of the invention for surface treatment, a major problem for all methods involving species within channels is the nonspecific adsorption of said species to the walls of said channels. This problem is particularly exacerbated in the case of channels of small dimensions and of biological macromolecules, the latter often being amphiphilic.
In the case of analytical methods, the consequence of this phenomenon of nonspecific adsorption to the walls by species contained in the sample or the fluid used to analyse said sample, is to delay certain analytes and to create an additional dispersion and therefore a loss of resolution. This adsorption may also give rise to a contamination of the channel walls, liable to affect the fluids intended to be subsequently introduced into this channel. Finally, if the analysis to be carried out on the species involves a specific interaction of the species with the separation medium, as in chromatography, electrochromatography or affinity electrophoresis methods, or with predetermined areas of the walls, as in hybridization methods such as “DNA chips” or “protein chips”, or else with solid walls contained in the channel or container, as in methods of separation by affinity with latexes, these adsorption phenomena may compete with the desired specific interactions and interfere with or prevent the analysis.
Another limitation, which concerns more particularly electrokinetic separation methods, is electroosmosis, an overall movement of the separation medium due to the presence of charges on the walls of the capillary or of the channel. Since this movement is often variable over time and is not uniform, it is harmful to the reproducibility of the measurements and to the resolution. It is due to the charges which may be present at the surface of the capillary on account of its chemical structure, but may also be generated or increased by the adsorption onto the wall of charged species initially contained in the samples to be separated, and in particular proteins.
The present invention is more particularly concerned with the inhibition of these two phenomena, namely adsorption of species to the surfaces and/or electroosmosis.
Methods have already been proposed for combating electroosmosis and/or adsorption of species to surfaces. A first type of method involves treating the surface of the channel by adsorption of essentially neutral species, prior to the actual separation (Wiktorowicz et al., Electrophoresis, 11, 769, 1990, Tsuji et al., J. Chromatogr. 594, 317 (1992)). It has also been proposed to adsorb surface agents with a charge which is opposite to that of the wall, to reinforce the adhesion by electrostatic interactions.
In fact, these methods reduce electroosmosis to a certain degree, but they are relatively ineffective in preventing the adsorption of complex species of high molecular mass, such as, for example, proteins.
A more effective solution consists in irreversibly grafting an essentially neutral polymeric layer, such as acrylamide or polyvinyl alcohol, onto the walls, as described, for example, in U.S. Pat. No. 4,680,201, or alternatively U.S. Pat. No. 5,502,169 or U.S. Pat. No. 5,112,460. Ready-to-use treated capillaries are thus commercially available. These irreversibly treated capillaries give good reduction of electroosmosis for a certain number of separations. Unfortunately, they have a limited life span and are expensive.
It has also been proposed to use, in the separation medium, polymers with properties of adsorption to walls, such as methylcellulose (Hjerten, Chromatographic reviews, 9, 122, 1967) or polyvinylpyrrolidone (Mazzeo at al., Anal. Chem., 63, 2852, 1991). In application WO 98/10274, copolymers having affinity with walls of silica and capable of significantly reducing electroosmosis are proposed. The polymers described are triblock polymers of low molecular masses (typically less than 20,000), of the polyoxyethylene-polyoxypropylene-polyoxypropylene (POE-POP-POE) family. However, these polymers have a limited range of application. They require a change in temperature between the introduction into the capillary and the analytical phase, they only exert their beneficial effect at high concentrations, and they are also relatively hydrophobic, which makes them unsuitable for example for DNA sequencing. In addition, in these various methods of the prior art using polymers in the separation medium, the presence of the polymer is accompanied by a considerable variation in the physical properties, and in particular by a considerable increase in viscosity, which may pose problems for the introduction of fluid into the channel and for the separation properties themselves.
In U.S. Pat. No. 5,552,028 already cited above, it is also proposed to use separation media comprising a sieving medium and a surface interaction component consisting of a polymer with properties of adsorption to walls, having a molecular mass of between 5,000 and 1,000,000, of the disubstituted acrylamide polymer type. These matrices, and more particularly polydimethylacrylamide (PDMA), make it possible to reduce electroosmosis and, for some applications, such as sequencing, produce good separation properties. However, they are relatively hydrophobic, which limits their effectiveness for some applications such as DNA sequencing, and is even more harmful for other applications such as protein separation. Moreover, they produce slow separations.
Consequently, although many methods have been proposed for reducing adsorption to walls and/or electroosmosis, they are not found to be totally satisfactory.